Site-resolved measurement of microsecond-to-millisecond conformational-exchange processes in proteins by solid-state NMR spectroscopy

Abstract

We demonstrate that conformational exchange processes in proteins on microsecond-to-millisecond time scales can be detected and quantified by solid-state NMR spectroscopy. We show two independent approaches that measure the effect of conformational exchange on transverse relaxation parameters, namely Carr-Purcell-Meiboom-Gill relaxation-dispersion experiments and measurement of differential multiple-quantum coherence decay. Long coherence lifetimes, as required for these experiments, are achieved by the use of highly deuterated samples and fast magic-angle spinning. The usefulness of the approaches is demonstrated by application to microcrystalline ubiquitin. We detect a conformational exchange process in a region of the protein for which dynamics have also been observed in solution. Interestingly, quantitative analysis of the data reveals that the exchange process is more than 1 order of magnitude slower than in solution, and this points to the impact of the crystalline environment on free energy barriers.

Figure 1

Numerical simulations of the differential decay rates of zero- and double-quantum coherences (differential multiple-quantum decay rate), ΔR_MQ = R_DQ – R_ZQ, in a ¹H–¹⁵N spin pair undergoing exchange. A two-site exchange system involving a major state (populated at 90%) and a minor state (10%) was assumed, with an exchange rate k_ex = k_AB + k_BA, where k_AB denotes the forward rate constant. ΔR_MQ is shown as a function of the exchange-rate constant. The different simulations assume either only isotropic chemical-shift modulation (Δν_N = 160 Hz, Δν_H = 800 Hz), only CSA/CSA modulations (jumps by 30°), or both, as indicated in the insert. Details about the simulation parameters and additional simulations are provided in the Supporting Information.

Figure 2

Measurement of differential multiple-quantum decay rates. (a) Pulse sequence used in this study. Differential zero- and double-quantum line broadening is obtained from separate experiments that probe the coherences, 2H_xN_x and 2H_yN_y, respectively, which are selected by setting the phases of the pulses at the end of the MQC evolution delay. Details about delays and phase settings are shown in Figure S6 in the Supporting Information. (b,c) Experimental data obtained on a microcrystalline sample of ubiquitin at 300 K: (b) Representative examples of the buildup of 2H_yN_y from 2H_xN_x, along with best-fit curves, ΔR_MQ = (2 atanh(⟨2H_yN_y⟩/⟨2H_xN_x⟩))/T. Error bars were obtained from 2 times the standard deviation of the spectral noise. (c) Fitted residue-wise differential multiple-quantum decay-rate constants ΔR_MQ, using three different relaxation delays. Error margins were determined from Monte Carlo simulation based on error bars determined from twice the spectral noise. Residues with particularly large ΔR_MQ are indicated. Note that, in principle, a single relaxation delay would suffice to determine ΔR_MQ. (d) Residues for which large ΔR_MQ are observed (I23, K27, T55) as well as unobservable resonances (E24, N25) in ¹H-detected HSQC-type spectra are plotted onto the structure. The H-bonding of I23(HN)-R54(CO) is indicated.

Figure 3

(a) Pulse sequence used in this study to measure ¹⁵N CPMG relaxation-dispersion data on deuterated proteins in the solid state. Details are shown in the Supporting Information. (b) ¹⁵N CPMG RD solid-state NMR data obtained on microcrystalline ubiquitin at 300 K sample temperature, collected at a ¹H Larmor frequency of 600 MHz (red) and 800 MHz (black). Note the different scale in the data of Ile23. Upper and lower error bars of R_2eff were determined from Monte Carlo simulations, based on twice the spectral noise (see Figure S17). For residues I23, K27, and T55, solid lines represent the Bloch–McConnell fit, in other cases lines represent the mean values of the individual data points. Data for all other residues are shown in the Supporting Information.

Figure 4

Structural comparison of the microcrystals used in this study, PDB 3ons(48) (a,b), and a solution structure of ubiquitin, PDB 1d3z(49) (c,d). Intramolecular H-bonding among backbone atoms and H-bonds involving side chains are indicated in red and blue. Water-mediated intermolecular H-bonds are shown in yellow. Neighboring molecules in the crystal lattice are shown in light blue in (a) and (b), highlighting residues K63 and E64 of the neighboring molecules. Note the different conformation of the loop E51-R54 in the two structures (a,b) and (c,d), resulting in a flip of the orientation of NH(G53) and CO(D52).